Chapter 7:Chapter 7:

Analysis of specific proteins & Analysis of specific proteins & protein qualityprotein quality

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Proteins are polymers of some 21 different amino acids joined together by peptide bonds. Because of the variety of side chains that occur when these amino acids are linked together, the different proteins may have different chemical properties and widely different secondary and tertiary structures

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The side chains may be polar or nonpolar. High levels of polar amino acid residues in a protein increase water solubility. The most polar side chains are those of the basic and acidic amino acids. These amino acids are present at high levels in the soluble albumins and globulins. In contrast, the wheat proteins, gliadin and glutenin, have low levels of polar side chains and are quite insoluble in water.

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PROTEIN CLASSIFICATIONPROTEIN CLASSIFICATION

Simple ProteinsSimple proteins yield only amino acids on hydrolysis and include the following

classes:• Albumins. Soluble in neutral, salt-free

water. Usually these are proteins of relatively low molecular weight. Examples are egg albumin, lactalbumin, and serum albumin in the whey proteins of milk, leucosin of cereals, and legumelin in legume seeds.

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Globulins. Soluble in neutral salt solutions and almost insoluble in water. Examples are serum globulins and (3-lactoglobulin in milk, myosin and actin in meat, and glycinin in soybeans.

• Glutelins. Soluble in very dilute acid or base and insoluble in neutral solvents. These proteins occur in cereals, such as glutenin in wheat and oryzenin in rice.

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Prolamins. Soluble in 50 to 90 percent ethanol and insoluble in water. These proteins have large amounts of proline and glutamic acid and occur in cereals. Examples are zein in corn, gliadin in wheat, and hordein in barley.

• Scleroproteins. Insoluble in water and neutral solvents and resistant to enzymic hydrolysis. These are fibrous proteins serving structural and binding purposes. Collagen of muscle tissue is included in this group, as is gelatin, which is derived from it. Other examples include elastin, a component of tendons, and keratin, component of hair and hoofs.

• Histories. Basic proteins, as defined by their high content of lysine and arginine. Soluble in water and precipitated by

ammonia. .

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Conjugated ProteinsConjugated ProteinsConjugated proteins contain an amino

acid part combined with a nonprotein material such as a lipid, nucleic acid, or carbohydrate. Some of the major conjugated proteins are as follows:

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• Phosphoproteins. An important group that includes many major food proteins, Phosphate groups are linked to the hydroxyl groups of serine and threonine,This group includes casein of milk and

the phosphoproteins of egg yolk.• Lipoproteins. These are combinations

of lipids with protein and have excellent emulsifying capacity. Lipoproteins occur in milk and egg yolk.

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• Nucleoproteins. These are combinations of nucleic acids with protein. These compounds are found in cell nuclei.

• Glycoproteins. These are combinations of carbohydrates with protein. Usually the amount of carbohydrate is small, but some glycoproteins have carbohydrate contents of 8 to 20 percent. An example of such a mucoprotein is ovomucin of egg white.

• Chromopmteins. These are proteins with a colored prosthetic group. There are many compounds of this type, including hemoglobin and myoglobin, chlorophyll, and flavoproteins.

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Derived ProteinsDerived ProteinsThese are compounds obtained by chemical

or enzymatic methods and are divided into primary and secondary derivatives, depending on the extent of change that has taken place. Primary derivatives are slightly modified and are insoluble in water; rennet coagulated casein is an example of a primary derivative. Secondary derivatives are more extensively changed and include proteoses, peptones, and peptides. The difference between these breakdown products is in size and solubility

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These breakdown products are formed during the processing of many foods, for example, during ripening of cheese.

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Protein structureProtein structure

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Analysis of specific proteins Analysis of specific proteins Separation techniques

◦precipitation◦adsorption

ion-exchange chromatography affinity chromatography

◦size dialysis ultrafiltration size-exclusion chromatography

◦electrophoresisNon separation techniques

◦immunoassay14

Use of precipitation in Use of precipitation in protein analysisprotein analysis

Often the first step in protein separation

Separated proteins can then be more easily identified and characterised

Exploits differences in solubility of different types of proteins to partially purify protein of interest before identification◦solubility determined by type and charge

on amino acid -R groups15

Proteins selectively precipitated by:◦salting out (ionic strength)

use of neutral salts such as ammonium sulphate [(NH4)2SO4], NaCl and KCl

exploits lower but differing solubility's of different protein in increasing concentration of neutral salt solutions

◦iso-electric precipitation iso-electric point, is pH at which protein has

no net charge exploiting different isoelectric points of

proteins16

Proteins selectively precipitated by:◦solvent fractionation

protein solubility at fixed pH and ionic strength is a function of the dielectric constant

exploiting generally lower but differing solubility of most protein in water miscible organic solvents (ethanol, acetone)

Organic solvents (5% to 60%) decrease the ionization charge of amino acids

◦denaturation exploits differential susceptibility of proteins

to heat & extreme pH denaturation leading to precipitation 17

Separation of proteins by Separation of proteins by sizesizeProteins MW from 10,000 - 1,000,000

Dalton sizeActual separation depends on the stokes

radius not on the molecular weight of the protein ◦ Stokes radius: average radius of protein in

solutionDialysis

◦ separation of proteins in a solution by selective diffusion through a semipermeable membrane

◦ protein extract placed in sealed dialysis tubing in large volume of buffer (X 500-1000) for 12 hr.

◦ small proteins leave bag larger proteins are retained 18

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Separation of proteins by Separation of proteins by sizesize

Micro, Ultra & Nanofiltration◦ differ in porosity & pressure◦ can separate proteins on basis of

size using semi-permeable membrane under applied pressure

◦ molecules larger than the membrane cut off are retained Micro (0.1 to 5µm): remove particles &

microorganisms Ultra (0.005 to 0.1µm): concentrate

and fractionate protein solution, remove salt

nano (0.0001 to 0.005µm): remove monovalent ions from salt whey

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Use of electrophoresis in Use of electrophoresis in protein analysisprotein analysis

Separation depends on friction and charge of the protein.

Mobility = (applied voltage) (net charge on molecule)friction of the molecule

◦ proteins are positively or negatively charged negatively charged when pH is above pI positively charged when when pH is below pI

Magnitude of protein charge and applied voltage will determine how far a protein will migrate

Mobility decreases with increased frication due to higher stokes radius

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Used to determine protein composition of a food product ◦ can identify how different food processing

techniques can alter protein composition of soy protein isolates or whey protein isolates used as food ingredients

◦ can identify species of meat used in foods◦ can determine purity of proteins eg enzymes

used for food processing◦ individual proteins identified by their

migration distance during electrophoresis◦ individual proteins quantified by the intensity

of staining of bands on electrophoresis gel22

The determination of the The determination of the amino acid composition of amino acid composition of proteinsproteins

Amino acid composition is used to:◦assess nutritional quality of protein◦characterise and identify a newly

isolated protein◦calculate nitrogen conversion factors

for specific proteinsFood hydrolysedReleased amino acids separated

chromatographicallyIndividual amino acids are

identified and quantified 23

Amino acid analysis - Amino acid analysis - hydrolysishydrolysisBoiling 6N HCl for 24 hr to release

amino acidsBut some amino acids destroyed:

◦typtophan completely◦methionine, cysteine, threonine and serine

progressively◦asparagine and glutamine converted to

aspartic & glutamic acids respectively-not measured

◦isoleucine and valine linked peptide bonds hydrolysed more slowly

◦tyrosine may be oxidised24

Amino acid analysis – hydrolysisAmino acid analysis – hydrolysisTyptophan - separately assayed

chromatographically after alkaline hydrolysis

Methionine and cysteine: separate chromatogram, hydrolysis in performic acid, give cysteic acid◦ HCL hydrolysis & chromatography

Threonine and serine – zero - time levels estimated by sampling at (24, 48 and 72hr) during hydrolysis◦ interpolating line to zero time.

Asparagine and glutamine converted to aspartic acid and glutamic acid respectively - cannot be measured

Tyrosine may be oxidised - difficult to control 25